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1.
Mol Cell ; 81(2): 370-385.e7, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33271062

RESUMEN

The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.


Asunto(s)
Proteína 5 Relacionada con la Autofagia/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , Intestino Grueso/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Animales Recién Nacidos , Proteína 5 Relacionada con la Autofagia/deficiencia , Caspasa 8/genética , Caspasa 8/metabolismo , Muerte Celular/genética , Proteína de Dominio de Muerte Asociada a Fas/deficiencia , Regulación de la Expresión Génica , Glucosa/antagonistas & inhibidores , Glucosa/farmacología , Células HEK293 , Células HT29 , Humanos , Intestino Grueso/efectos de los fármacos , Intestino Grueso/patología , Células Jurkat , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Metformina/antagonistas & inhibidores , Metformina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Transducción de Señal , Sirolimus/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo
2.
Mol Cell ; 75(3): 457-468.e4, 2019 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-31230815

RESUMEN

Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point of input from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressant TAM kinases are promoters of pro-inflammatory necroptosis, shedding light on the biological complexity of the regulation of inflammation.


Asunto(s)
Proteínas Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Síndrome de Respuesta Inflamatoria Sistémica/genética , Tirosina Quinasa c-Mer/genética , Animales , Apoptosis/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Necroptosis/genética , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Síndrome de Respuesta Inflamatoria Sistémica/patología , Factor de Necrosis Tumoral alfa/genética , Tirosina Quinasa del Receptor Axl
3.
Nucleic Acids Res ; 48(6): 2956-2968, 2020 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-32025719

RESUMEN

Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues in histones H3 and H4, marks that are generally associated with transcriptional repression. However, we found that PRMT5 inhibition or depletion led to more genes being downregulated than upregulated, indicating that PRMT5 can also act as a transcriptional activator. Indeed, the global level of histone H3K27me3 increases in PRMT5 deficient cells. Although PRMT5 does not directly affect PRC2 enzymatic activity, methylation of histone H3 by PRMT5 abrogates its subsequent methylation by PRC2. Treating AML cells with an EZH2 inhibitor partially restored the expression of approximately 50% of the genes that are initially downregulated by PRMT5 inhibition, suggesting that the increased H3K27me3 could directly or indirectly contribute to the transcription repression of these genes. Indeed, ChIP-sequencing analysis confirmed an increase in the H3K27me3 level at the promoter region of a quarter of these genes in PRMT5-inhibited cells. Interestingly, the anti-proliferative effect of PRMT5 inhibition was also partially rescued by treatment with an EZH2 inhibitor in several leukemia cell lines. Thus, PRMT5-mediated crosstalk between histone marks contributes to its functional effects.


Asunto(s)
Arginina/metabolismo , Histonas/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Transcripción Genética , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Eliminación de Gen , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Metilación , Ratones Noqueados , Modelos Biológicos , Nucleosomas/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores
4.
PLoS Biol ; 16(8): e2005756, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30157175

RESUMEN

Necroptosis is a lytic programmed cell death mediated by the RIPK1-RIPK3-MLKL pathway. The loss of Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression and necroptotic potential have been previously reported in several cancer cell lines; however, the extent of this loss across cancer types, as well as its mutational drivers, were unknown. Here, we show that RIPK3 expression loss occurs progressively during tumor growth both in patient tumor biopsies and tumor xenograft models. Using a cell-based necroptosis sensitivity screen of 941 cancer cell lines, we find that escape from necroptosis is prevalent across cancer types, with an incidence rate of 83%. Genome-wide bioinformatics analysis of this differential necroptosis sensitivity data in the context of differential gene expression and mutation data across the cell lines identified various factors that correlate with resistance to necroptosis and loss of RIPK3 expression, including oncogenes BRAF and AXL. Inhibition of these oncogenes can rescue the RIPK3 expression loss and regain of necroptosis sensitivity. This genome-wide analysis also identifies that the loss of RIPK3 expression is the primary factor correlating with escape from necroptosis. Thus, we conclude that necroptosis resistance of cancer cells is common and is oncogene driven, suggesting that escape from necroptosis could be a potential hallmark of cancer, similar to escape from apoptosis.


Asunto(s)
Proteínas Proto-Oncogénicas B-raf/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Necrosis/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor Axl
5.
Proc Natl Acad Sci U S A ; 114(45): 11944-11949, 2017 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-29078411

RESUMEN

Apoptosis and necroptosis are two distinct cell death mechanisms that may be activated in cells on stimulation by TNFα. It is still unclear, however, how apoptosis and necroptosis may be differentially regulated. Here we screened for E3 ubiquitin ligases that could mediate necroptosis. We found that deficiency of Pellino 1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner during necroptosis. Ubiquitination of RIPK1 by PELI1 promotes the formation of necrosome and execution of necroptosis. Although PELI1 is not directly involved in mediating the activation of RIPK1, it is indispensable for promoting the binding of activated RIPK1 with its downstream mediator RIPK3 to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in necroptosis. However, we show that PELI1 deficiency sensitizes cells to both RIPK1-dependent and RIPK1-independent apoptosis as a result of down-regulated expression of c-FLIP, an inhibitor of caspase-8. Finally, we show that Peli1-/- mice are sensitized to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of RIPK1 that differentially controls the activation of necroptosis and apoptosis.


Asunto(s)
Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Necrosis/genética , Proteínas Nucleares/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
6.
Immunology ; 150(4): 379-388, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-27936492

RESUMEN

The nuclear factor-κB (NF-κB) family of transcription factors play an essential role for the regulation of inflammatory responses, immune function and malignant transformation. Aberrant activity of this signalling pathway may lead to inflammation, autoimmune diseases and oncogenesis. Over the last two decades great progress has been made in the understanding of NF-κB activation and how the response is counteracted for maintaining tissue homeostasis. Therapeutic targeting of this pathway has largely remained ineffective due to the widespread role of this vital pathway and the lack of specificity of the therapies currently available. Besides regulatory proteins and microRNAs, long non-coding RNA (lncRNA) is emerging as another critical layer of the intricate modulatory architecture for the control of the NF-κB signalling circuit. In this paper we focus on recent progress concerning lncRNA-mediated modulation of the NF-κB pathway, and evaluate the potential therapeutic uses and challenges of using lncRNAs that regulate NF-κB activity.


Asunto(s)
Transformación Celular Neoplásica/inmunología , Inflamación/inmunología , FN-kappa B/metabolismo , ARN Largo no Codificante/genética , Transducción de Señal , Animales , Regulación de la Expresión Génica , Terapia Genética , Homeostasis , Humanos , Inflamación/terapia , Ratones , Terapia Molecular Dirigida , FN-kappa B/genética , ARN Largo no Codificante/uso terapéutico
7.
Nat Commun ; 15(1): 3415, 2024 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-38649367

RESUMEN

An important epigenetic component of tyrosine kinase signaling is the phosphorylation of histones, and epigenetic readers, writers, and erasers. Phosphorylation of protein arginine methyltransferases (PRMTs), have been shown to enhance and impair their enzymatic activity. In this study, we show that the hyperactivation of Janus kinase 2 (JAK2) by the V617F mutation phosphorylates tyrosine residues (Y149 and Y334) in coactivator-associated arginine methyltransferase 1 (CARM1), an important target in hematologic malignancies, increasing its methyltransferase activity and altering its target specificity. While non-phosphorylatable CARM1 methylates some established substrates (e.g. BAF155 and PABP1), only phospho-CARM1 methylates the RUNX1 transcription factor, on R223 and R319. Furthermore, cells expressing non-phosphorylatable CARM1 have impaired cell-cycle progression and increased apoptosis, compared to cells expressing phosphorylatable, wild-type CARM1, with reduced expression of genes associated with G2/M cell cycle progression and anti-apoptosis. The presence of the JAK2-V617F mutant kinase renders acute myeloid leukemia (AML) cells less sensitive to CARM1 inhibition, and we show that the dual targeting of JAK2 and CARM1 is more effective than monotherapy in AML cells expressing phospho-CARM1. Thus, the phosphorylation of CARM1 by hyperactivated JAK2 regulates its methyltransferase activity, helps select its substrates, and is required for the maximal proliferation of malignant myeloid cells.


Asunto(s)
Apoptosis , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Janus Quinasa 2 , Proteína-Arginina N-Metiltransferasas , Tirosina , Humanos , Fosforilación , Janus Quinasa 2/metabolismo , Janus Quinasa 2/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Tirosina/metabolismo , Línea Celular Tumoral , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Metilación , Especificidad por Sustrato , Células HEK293 , Ciclo Celular , Mutación
8.
J Clin Invest ; 133(13)2023 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-37200093

RESUMEN

During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation, which can cause life-threatening diseases and cancer. Here, we identify a role of double PHD fingers 2 (DPF2) in modulating inflammation. DPF2 is a defining subunit of the hematopoiesis-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncovered that hematopoiesis-specific Dpf2-KO mice developed leukopenia, severe anemia, and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration resembling a clinical hyperinflammatory state. Dpf2 loss impaired the polarization of macrophages responsible for tissue repair, induced the unrestrained activation of Th cells, and generated an emergency-like state of HSC hyperproliferation and myeloid cell-biased differentiation. Mechanistically, Dpf2 deficiency resulted in the loss of the BAF catalytic subunit BRG1 from nuclear factor erythroid 2-like 2-controlled (NRF2-controlled) enhancers, impairing the antioxidant and antiinflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of NRF2 suppressed the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes an essential role of the DPF2-BAF complex in licensing NRF2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.


Asunto(s)
Cromatina , Neoplasias , Ratones , Animales , Antioxidantes , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Ensamble y Desensamble de Cromatina , Inflamación/genética , Expresión Génica , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
9.
Front Pharmacol ; 9: 218, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29615902

RESUMEN

Glioblastoma multiforme (GBM) is the most malignant primary adult brain tumor. The current standard of care is surgical resection, radiation, and chemotherapy treatment, which extends life in most cases. Unfortunately, tumor recurrence is nearly universal and patients with recurrent glioblastoma typically survive <1 year. Therefore, new therapies and therapeutic combinations need to be developed that can be quickly approved for use in patients. However, in order to gain approval, therapies need to be safe as well as effective. One possible means of attaining rapid approval is repurposing FDA approved compounds for GBM therapy. However, candidate compounds must be able to penetrate the blood-brain barrier (BBB) and therefore a selection process has to be implemented to identify such compounds that can eliminate GBM tumor expansion. We review here psychiatric and non-psychiatric compounds that may be effective in GBM, as well as potential drugs targeting cell death pathways. We also discuss the potential of data-driven computational approaches to identify compounds that induce cell death in GBM cells, enabled by large reference databases such as the Library of Integrated Network Cell Signatures (LINCS). Finally, we argue that identifying pathways dysregulated in GBM in a patient specific manner is essential for effective repurposing in GBM and other gliomas.

10.
Nat Cell Biol ; 20(1): 58-68, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29203883

RESUMEN

Ubiquitylation of the TNFR1 signalling complex (TNF-RSC) controls the activation of RIPK1, a kinase critically involved in mediating multiple TNFα-activated deleterious events. However, the molecular mechanism that coordinates different types of ubiquitylation modification to regulate the activation of RIPK1 kinase remains unclear. Here, we show that ABIN-1/NAF-1, a ubiquitin-binding protein, is recruited rapidly into TNF-RSC in a manner dependent on the Met1-ubiquitylating complex LUBAC to regulate the recruitment of A20 to control Lys63 deubiquitylation of RIPK1. ABIN-1 deficiency reduces the recruitment of A20 and licenses cells to die through necroptosis by promoting Lys63 ubiquitylation and activation of RIPK1 with TNFα stimulation under conditions that would otherwise exclusively activate apoptosis in wild-type cells. Inhibition of RIPK1 kinase and RIPK3 deficiency block the embryonic lethality of Abin-1 -/- mice. We propose that ABIN-1 provides a critical link between Met1 ubiquitylation mediated by the LUBAC complex and Lys63 deubiquitylation by phospho-A20 to modulate the activation of RIPK1.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Fibroblastos/metabolismo , Fosfoproteínas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Apoptosis/genética , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Transformada , Fibroblastos/citología , Regulación de la Expresión Génica , Genes Letales , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación
11.
Cell Rep ; 24(10): 2643-2657, 2018 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-30184499

RESUMEN

Protein arginine methyltransferase 5 (PRMT5) is overexpressed in many cancer types and is a promising therapeutic target for several of them, including leukemia and lymphoma. However, we and others have reported that PRMT5 is essential for normal physiology. This dependence may become dose limiting in a therapeutic setting, warranting the search for combinatorial approaches. Here, we report that PRMT5 depletion or inhibition impairs homologous recombination (HR) DNA repair, leading to DNA-damage accumulation, p53 activation, cell-cycle arrest, and cell death. PRMT5 symmetrically dimethylates histone and non-histone substrates, including several components of the RNA splicing machinery. We find that PRMT5 depletion or inhibition induces aberrant splicing of the multifunctional histone-modifying and DNA-repair factor TIP60/KAT5, which selectively affects its lysine acetyltransferase activity and leads to impaired HR. As HR deficiency sensitizes cells to PARP inhibitors, we demonstrate here that PRMT5 and PARP inhibitors have synergistic effects on acute myeloid leukemia cells.


Asunto(s)
Proteína-Arginina N-Metiltransferasas/metabolismo , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/fisiología , Muerte Celular , Línea Celular Tumoral , Reparación del ADN/genética , Reparación del ADN/fisiología , Código de Histonas/genética , Código de Histonas/fisiología , Histonas/metabolismo , Humanos , Lisina Acetiltransferasa 5/genética , Lisina Acetiltransferasa 5/metabolismo , Lisina Acetiltransferasas/genética , Lisina Acetiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética
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